The invention relates to p-thienylbenzylamides of formula (I) 
in which R(1), R(2), R(3), R(4), R(5), R(6) and X have the meanings given below. The compounds of formula (I) are potent agonists of angiotensin-(1-7) receptors. As a result of the stimulation of these receptors, which is elicited by the compounds of formula (I), and the production and release of the vasorelaxing and cardioprotective messengers cyclic guanosine monophosphate (cGMP) and nitrogen monoxide (NO) associated with endothelial cells, the compounds of formula (I) are suitable as pharmaceutically active compounds for treating and preventing hypertension; cardiac hypertrophy; cardiac insufficiency; coronary heart diseases, such as angina pectoris; and endothelial dysfunction or endothelial damage, for example, as a consequence of atherosclerotic processes or in association with diabetes mellitus.
PCT application WO-0068226 describes 1-(p-thienylbenzyl)imidazoles as agonists of angiotensin-(1-7) receptors for the treatment and/or prophylaxis of hypertension, cardiac hypertrophy, cardiac insufficiency and endothelial dysfunction or endothelial damage.
In view of the multifarious possibilities for using angiotensin (ANG)-(1-7) receptor agonists as pharmaceuticals, and the demands for such properties, there is a need for further ANG-(1-7) receptor agonists which exhibit favorable activity and selectivity (i.e., a good pharmacodynamic or pharmacokinetic profile).
It has been found, surprisingly, that p-thienylbenzylamides of formula (I) have a pronounced effect on angiotensin-(1-7) receptors and mimic the biological effect of the effector hormone angiotensin-(1-7).
One part of the subject-matter of the invention consequently relates to compounds of formula (I) 
in which the indicated radicals have the following meanings:
R(1) is chosen from among
1. (C1-C5)-alkyl, unsubstituted or substituted by a radical chosen from among NH2, halogen, Oxe2x80x94(C1-C3)-alkyl, COxe2x80x94Oxe2x80x94(C1-C3)-alkyl and CO2H;
2. (C3-C8)-cycloalkyl;
3. (C1-C3)-alkyl-(C3-C8)-cycloalkyl;
4. (C6-C10)-aryl, unsubstituted or substituted by a radical chosen from halogen and Oxe2x80x94(C1-C3)-alkyl;
5. (C1-C3)-alkyl-(C6-C10)-aryl, where the aryl radical is unsubstituted or substituted by a radical chosen from halogen and Oxe2x80x94(C1-C3)-alkyl;
6. (C1-C5)-heteroaryl; and
7. (C1-C3)-alkyl-(C1-C5)-heteroaryl;
R(2) is chosen from among
1. hydrogen;
2. (C1-C6)-alkyl, unsubstituted or substituted by a radical chosen from halogen and Oxe2x80x94(C1-C3)-alkyl;
3. (C3-C8)-cycloalkyl;
4. (C1-C3)-alkyl-(C3-C8)-cycloalkyl;
5. (C6-C10)-aryl, unsubstituted or substituted by a radical chosen from among halogen, Oxe2x80x94(C1-C3)-alkyl and COxe2x80x94Oxe2x80x94(C1-C3)-alkyl; and
6. (C1-C3)-alkyl-(C6-C10)-aryl, unsubstituted or substituted by a radical chosen from halogen and Oxe2x80x94(C1-C3)-alkyl;
R(3) is chosen from among
1. hydrogen;
2. COOH; and
3. COOxe2x80x94(C1-C4)-alkyl;
R(4) is chosen from among
1. hydrogen;
2. halogen; and
3. (C1-C4)-alkyl;
R(5) is chosen from among
1. hydrogen, and
2. (C1-C6)-alkyl;
R(6) is chosen from among
1. hydrogen;
2. (C1-C6)-alkyl;
3. (C1-C3)-alkyl-(C3-C8)-cycloalkyl; and
4. (C2-C6)-alkenyl;
X is chosen from among
1. oxygen, and
2. NH;
in all the stereoisomeric forms thereof, and mixtures thereof in all ratios, and the physiologically tolerated salts thereof.
Unless otherwise indicated, the term alkyl encompasses straight-chain or branched saturated hydrocarbon radicals. This also applies to substituents which are derived therefrom, such as alkoxy or the radicals SO2NHCOO-alkyl and SO2NHCONH-alkyl. Examples of alkyl radicals include methyl, ethyl, n-propyl, isopropyl, isobutyl, n-butyl, n-hexyl, isohexyl and n-heptyl. Examples of alkoxy radicals include methoxy, ethoxy and propoxy, such as n-propoxy and isopropoxy.
Alkenyl denotes singly or multiply unsaturated hydrocarbon radicals in which the double bonds can be present in any arbitrary position. Examples of alkenyl radicals include vinyl, prop-2-enyl (allyl), prop-1-enyl and butenyl.
Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
Halogen denotes fluorine, chlorine, bromine or iodine, and in one embodiment, the halogen is chlorine or fluorine.
Examples of aryl radicals include phenyl and naphthyl (1- or 2-naphthyl).
In substituted aryl radicals,.the substituents can be located in any positions in relation to each other.
Heteroaryl is understood as meaning radicals of monocyclic 5-membered or 6-membered aromatic ring systems. They can be regarded as being radicals which are derived from cyclopentadienyl and phenyl by the replacement of one or two CH groups and/or CH2 groups with S, O, N or NH (or N carrying a substituent, such as Nxe2x80x94CH3), in connection with which the aromatic ring system is preserved or an aromatic ring system is formed. In addition to the one, two, three or four ring heteroatoms, they may contain one, two, three, four or five ring carbon atoms ((C1-C5)-heteroaryl). Examples of suitable heteroaryls include furanyl, thienyl, pyrrolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrazinyl and pyrimidyl. A heteroaryl radical can be bonded by way of any suitable carbon atom.
In one embodiment,
R(1) is
1. (C1-C5)-alkyl, such as methyl; or
2. (C1-C5)-alkyl, substituted by a radical chosen from COxe2x80x94Oxe2x80x94(C1-C3)-alkyl and CO2H; such as carboxypropionyl and methoxycarbonylpropionyl; or
3. (C1-C3)-alkyl-(C3-C8)-cycloalkyl, such as cyclohexylmethyl; or
4. (C6-C10)-aryl, such as phenyl; or
5. (C1-C5)-heteroaryl, such as furanoyl.
R(2) is
1. hydrogen; or
2. (C1-C6)-alkyl, such as isopropyl; or
3. (C1-C6)-alkyl, substituted by Oxe2x80x94(C1-C3)-alkyl, such as methoxymethylene; or
4. (C3-C8)-cycloalkyl, such as cyclopropyl and cyclohexyl; or
5. (C6-C10)-aryl, such as phenyl.
R(3) is
1. COOH; or
2. COOxe2x80x94(C1-C4)-alkyl, such as methoxycarbonyl and ethoxycarbonyl.
R(4) is hydrogen.
R(5) is (C1-C6)-alkyl, such as isobutyl.
R(6) is (C1-C6)-alkyl, such as methyl, ethyl or butyl.
The present invention encompasses all the stereoisomeric forms of the compounds of formula (I). In the compounds of formula (I) which contain centers of asymmetry, all these centers can, independently of each other, have the S or R configuration. The invention includes all the possible enantiomers and diastereomers, as well as mixtures of two or more diastereomeric forms, for example mixtures composed of enantiomers and/or diastereomers, in all ratios. When a cis/trans isomerism is present, both the cis and the trans form, and mixtures of these forms in all ratios, are part of the subject-matter of the invention. The invention also encompasses all the tautomeric forms of the compounds of formula (I).
Physiologically tolerated salts of compounds of formula (I) are understood as being both their inorganic salts and their organic salts, as described in Remington""s Pharmaceutical Sciences (A. R. Gennard, Editor, Mack Publishing Co, Easton Pa., 17th edition, pages 14-18, 1985). Because of their physiological and chemical stability and solubility, useful salts include, inter alia, sodium, potassium, calcium, magnesium and ammonium salts for acidic groups. Reactions of compounds of formula (I) with bases for the purpose of preparing the salts are, in general, carried out in accordance with customary procedures in a solvent or diluent.
The present invention furthermore encompasses solvates of compounds of formula (I), for example, hydrates or adducts with alcohols; and also derivatives of the compounds of formula (I), such as esters; and prodrugs and active metabolites.
In another embodiment, the compounds of formula (I) include those in which
R(1) is chosen from among
1. (C1-C5)-alkyl, unsubstituted or substituted by a radical chosen from among NH2, halogen, Oxe2x80x94(C1-C3)-alkyl, COxe2x80x94Oxe2x80x94(C1-C3)-alkyl and CO2H;
2. (C3-C6)-cycloalkyl;
3. (C1-C3)-alkyl-(C3-C6)-cycloalkyl;
4. (C6-C10)-aryl, unsubstituted. or substituted by a radical chosen from halogen and Oxe2x80x94(C1-C3)-alkyl;
5. (C1-C3)-alkyl-(C6-C10)-aryl, where the aryl radical is unsubstituted or substituted by a radical chosen from halogen and Oxe2x80x94(C1-C3)-alkyl;
6. (C3-C5)-heteroaryl; and
7. (C1-C3)-alkyl-(C3-C5)-heteroaryl;
R(2) is chosen from among
1. hydrogen;
2. (C1-C6)-alkyl, unsubstituted or substituted by a radical chosen from halogen and Oxe2x80x94(C1-C3)-alkyl;
3. (C3-C6)-cycloalkyl;
4. (C1-C3)-alkyl-(C3-C6)-cycloalkyl;
5. (C6-C10)-aryl, unsubstituted or substituted by a radical chosen from among halogen, Oxe2x80x94(C1-C3)-alkyl and COxe2x80x94Oxe2x80x94(C1-C3)-alkyl; and
6. (C1-C3)-alkyl-(C6-C10)-aryl, unsubstituted or substituted by a radical chosen from halogen and Oxe2x80x94(C1-C3)-alkyl;
R(3) is chosen from among
1. hydrogen;
2. COOH; and
3. COOxe2x80x94(C1-C4)-alkyl;
R(4) is chosen from among
1. hydrogen;
2. halogen; and
3. (C1-C4)-alkyl;
R(5) is chosen from
1. hydrogen, and
2. (C1-C4)-alkyl;
R(6) is chosen from among
1. hydrogen;
2. (C1-C4)-alkyl;
3. (C1-C3)-alkyl-(C3-C6)-cycloalkyl; and
4. (C3-C5)-alkenyl;
X is chosen from
1. oxygen,and
2. NH;
in all the stereoisomeric forms thereof, and mixtures thereof in all ratios, and the physiologically tolerated salts thereof.
In yet another embodiment, the compounds of formula (I) include those in which
R(1) is chosen from among
1. (C1-C3)-alkyl, unsubstituted or substituted by a radical chosen from among fluorine, methoxy, ethoxy, COxe2x80x94Oxe2x80x94(C1-C3)-alkyl and CO2H;
2. (C1-C3)-alkyl-cyclohexyl;
3. phenyl, substituted or unsubstituted by a radical chosen from fluorine and methoxy;
4. (C1-C3)-alkyl-phenyl, where the phenyl radical is unsubstituted or substituted by a radical chosen from fluorine and methoxy; and
5. furanyl, thienyl or pyridyl;
R(2) is chosen from among
1. hydrogen;
2. (C1-C6)-alkyl, unsubstituted or substituted by a radical chosen from among fluorine, methoxy and ethoxy;
3. phenyl, unsubstituted or substituted by a radical chosen from fluorine and methoxy; and
4. (C1-C6)-cycloalkyl;
R(4) is chosen from among
1. hydrogen;
2. methyl; and
3. chlorine;
R(5) is (C1-C4)-alkyl;
R(6) is (C1-C4)-alkyl;
and the radicals R(3) and X are as defined above, in all the stereoisomeric forms thereof, and mixtures thereof, and the physiologically tolerated salts thereof.
In an alternative embodiment, the compounds of formula (I) include those in which
R(1) is chosen from among
1. (C1-C3)-alkyl, unsubstituted or substituted by a radical chosen from among fluorine, methoxy, ethoxy, COxe2x80x94Oxe2x80x94(C1-C3)-alkyl and CO2H;
2. (C1-C3)-alkyl-cyclohexyl;
3. phenyl, substituted or unsubstituted by a radical chosen from fluorine and methoxy;
4. (C1-C3)-alkyl-phenyl, where the phenyl radical is unsubstituted or substituted by a radical chosen from fluorine and methoxy; and
5. furanyl, thienyl or pyridyl;
R(2) is chosen from among
1. hydrogen;
2. (C1-C6)-alkyl, unsubstituted or substituted by a radical chosen from among fluorine, methoxy and ethoxy;
3. phenyl, unsubstituted or substituted by a radical chosen from fluorine and methoxy; and
4. cyclopropyl or cyclohexyl;
R(4) is chosen from among
1. hydrogen;
2. methyl; and
3. chlorine;
R(5) is chosen from propyl and butyl, such as n-propyl, isopropyl and 2-isobutyl;
R(6) is chosen from among methyl, ethyl, n-propyl, isopropyl, n-butyl and isobutyl;
and the radicals R(3) and X are as defined above, in all the stereoisomeric forms thereof, and mixtures thereof, and the physiologically tolerated salts thereof.
In another embodiment, the compounds of formula (II) 
include those in which the radicals R(1), R(2), R(3), R(6) and X possess the above mentioned meanings, in all the stereoisomeric forms thereof, and mixtures thereof, and the physiologically tolerated salts thereof.
The invention furthermore relates to processes for preparing the compounds of formula (I), which processes are characterized by the reaction steps which are given below:
a) thiophene-3-boronic acids of formula (III), 
in which R(5) has the above mentioned meanings, and whose preparation is disclosed in EP-A 512 675, are reacted with p-bromobenzaldehydes of formula (IV) 
in which R(4) is as defined above, to give compounds of formula (V) 
in which R(4) and (R5) have the above mentioned meanings. This Suzuki-type cross-coupling reaction may be effected using palladium(II) acetate and triphenylphosphine or tetrakis(triphenylphosphine)palladium as catalysts in the presence of a base, such as cesium carbonate or potassium carbonate, for example in solvent mixtures composed of ethanol and toluene, at temperatures up to the boiling point of the solvents; corresponding reactions are described, for example, in Synthetic Commun. (1981) 11:513, J. Med. Chem. (1995) 38:2357-2377, and Liebigs Ann. (1995) 1253-1257, each of which is herein incorporated by reference.
b) The compounds of formula (V) can be converted, using primary amino compounds of formula (VI), 
in which R(2) and R(3) are defined as above, into compounds of formula (VII), 
in which the radicals R(2), R(3), R(4) and R(5) have the above mentioned meanings. This reductive amination may be effected by reacting the amines of formula (VI) with the aldehydes of formula (V) in an inert solvent, such as THF, in the presence of a reducing agent, such as sodium cyanoborohydride, and molecular sieve as a dehydrating agent, typically at room temperature or else at temperatures up to the boiling point of the solvent employed; corresponding reactions are described, for example, in Synthesis (1975) 135ff. As well as NaCNBH3, it is also alternatively possible to use, for example, lithium aluminum hydride LiAlH4, sodium borohydride NaBH4, sodium triacetoxyborohydride NaBH(OAc)3 or H2, Pd/C as reducing agents for this amination; corresponding reactions are described, for example, in Tetrahedron Lett. (1987) 28:749ff, Synthesis (1996) 11:1325-1330, and Tetrahedron Lett. (1982) 23:1929ff, each of which is herein incorporated by reference.
c) Acylating the compounds of formula (VII) with acyl chlorides of the R(1)xe2x80x94COCl type, in which R(1) is as defined above, results in amides of formula (VIII), 
in which the radicals R(1), R(2), R(3), R(4) and R(5) have the above mentioned meanings. This acylation is effected, in accordance with known methods, by reacting the compounds of formula (VII) with carbonyl chlorides (which are commercially available or can be obtained from the corresponding carboxylic acids by treating them with thionyl chloride) in an inert organic solvent, such as CH2Cl2, which is heated to reflux, in the presence of an organic or inorganic base.
d) The compounds of formula (VIII) are converted into the sulfonamides of formula (IX), 
in which R(1), R(2), R(3), R(4) and R(5) are defined as above, by eliminating the tert-butyl protecting group. This elimination may be effected by treating the compounds of formula (VIII) with organic acids, such as concentrated trifluoroacetic acid, in the presence of anisole.
e) The sulfonylurethanes of formula (Ia), 
in which R(1), R(2), R(3), R(4), R(5), and R(6) are defined as above, can be prepared from the sulfonamides of formula (IX) by reacting the latter with R(6)-substituted chloroformic esters in which R(6) is as described above. This reaction is effected in the presence of a base, such as pyridine, and of an acylation accelerator, such as 4-pyrrolidinopyridine, at temperatures of from room temperature (RT) to 150xc2x0 C., but typically at RT.
f) The sulfonylureas of formula (Ib), 
in which (R1), R(2), R(3), R(4), R(5), and R(6) are defined as above, can be obtained from the sulfonamides of formula (IX) by treating them with (R6)-substituted isocyanates in which R(6) is as described above. The reaction with the R(6)-substituted isocyanates is effected in the presence of a base in an inert solvent at temperatures of from RT to 150xc2x0 C.
Examples of suitable bases include alkali metal or alkaline earth metal hydroxides, hydrides, amides or alkoxides, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium hydride, potassium hydride, calcium hydride, sodium amide, potassium amide, sodium methoxide, sodium ethoxide and potassium tert-butoxide. Suitable inert solvents include ethers, such as THF, dioxane, ethylene glycol dimethyl ether or diglymes; ketones, such as acetone or butanone; nitriles, such as acetonitrile; nitro compounds, such as nitromethane; esters, such as ethyl acetate; amides, such as DMF N-methylpyrrolidone, or hexamethylphosphoric triamide; sulfoxides, such as DMSO; and hydrocarbons, such as benzene, toluene or xylenes. In addition, mixtures of these solvents with each other are also suitable.
The sulfonylureas of formula (Ib) can also be prepared by reacting amines R(6)xe2x80x94NH2 with sulfonyl isocyanate derivatives which are obtained from the sulfonamides of formula (IX), for example, by treating them with phosgene or a phosgene replacement (e.g., triphosgene) in accordance with methods which are known to the skilled person. Alternatively, the sulfonylureas of formula (Ib) can be prepared by reacting the sulfonamides of formula (IX) with 2,2,2-trichloroacetamide derivatives of a suitable amine R(6)xe2x80x94NH2 in the presence of a base in an inert, high-boiling solvent, such as DMSO. Additionally, the sulfonylureas of formula (Ib) can be prepared from the corresponding sulfonylurethane of formula (Ia) by reaction with ethyl chloroformate, under the action of the corresponding amine R(6)xe2x80x94NH2 in an inert, high-boiling solvent, such as toluene, at temperatures up to the boiling point of the respective solvent. Such a process is described, for example, in J. Med. Chem. (1995) 38:2357-2377, and in Bioorg. Med. Chem. (1997) 5:673-678, each of which is herein incorporated by reference. The N-unsubstituted sulfonylureas of formula (Ib), in which R(6) is hydrogen, are prepared, typically at temperatures of xe2x88x9210 to 0xc2x0 C., by using sulfuric acid to hydrolyze the sulfonamidonitriles (which result from the reaction of the sulfonamides of formula (IX) with cyanogen bromide in the presence of K2CO3 in acetonitrile).
The corresponding carboxylic acids of formula (I) can then be prepared, in accordance with known methods, as are described in the literature (for example in the standard works such as Houben-Weyl, Methoden der Organischen Chemie [Methods of Organic Chemistry], Georg Thieme Verlag; Stuttgart, Organic Reactions, John Wiley and Sons, Inc., New York; or Larock, Comprehensive Organic Transformations, VCH, Weinheim) by alkaline hydrolysis of the ester groups in the compounds of formula (I).
The vascular endothelium is a metabolically active organ which has a large number of regulatory functions and which is capable of synthesizing and releasing vasoactive substances. The pathogenesis of a variety of cardiovascular diseases, such as atherosclerosis and hypertension, correlates with a dysfunction in the blood vessel-lining endothelial layer (Eur. J. Clin. Invest. (1993) 23:670-685). An endothelial dysfunction is characterized by a reduced synthesis and/or release of the vasorelaxing, vasoprotective and antithrombotic and antiproliferative active messengers NO and cGMP, which play an essential role in the prevention and regression of vascular remodeling and arterial hypertension. Substances which are able to stimulate the synthesis and release of these messengers are therefore potentially valuable pharmaceuticals for treating all the diseases which are characterized by an endothelial dysfunction.
A large number of published experiments have verified the fact that a breakdown product of the renin-angiotensin system, i.e. the heptapeptide angiotensin-(1-7), is a potent endogenous effector hormone of the renin-angiotensin system (Hypertension (1991) 18[Suppl. III]:III126-III133), the biological effect of which hormone is elicited via the stimulation of specific receptors which preferentially bind angiotensin-(1-7) (Peptides (1993) 14:679-684, Hypertension (1997) 29[part 2]:388-393)). In many cases, this effect is directed against that of the vasoconstrictor hormone angiotensin II or is opposed to this effect in a counter-regulatory manner (Hypertension (1997) 30[part 2]: 535-541, Regulatory Peptides (1998) 78:13-18).
In Hypertension (1992) 19[suppl. II]:II49-II55 and in Am. J. Cardiol. (1998) 82:17S-19S, it was demonstrated that angiotensin-(1-7) stimulates the production and/or the release of NO/cGMP and of the prostaglandins E2 and I2, an effect which is not blocked by pretreatment with AT1 receptor and AT2 receptor antagonists.
Hypertension (1996) 27[part 2]:523-528, reported that angiotensin-(1-7) caused an endothelial-dependent relaxation in the intact coronary arteries of dogs and pigs, while J. Cardiovasc. Pharmacol. (1997) 30:676-682, reported that angiotensin-(1-7) caused an endothelium-dependent relaxation of intact rat aortas which had been previously contracted with KCl, with this relaxation not being affected by AT1 receptor antagonists.
Peptides (1993) 14:679-684 and Am. J. Physiol. (1995) 269:H313-H319, demonstrated that, when continuously infused through an osmotic minipump, angiotensin-(1-7) had a hypotensive effect in spontaneously hypertensive rats, with the same dose of angiotensin-(1-7) having no effect on blood pressure in normotensive rats. As a complement to these investigations, it was demonstrated, in Hypertension (1998) 31:699-705, that infusion of an angiotensin-(1-7) antibody increased the average arterial blood pressure in conscious, spontaneously hypertensive rats which had been pretreated with lisinopril and losartan.
In Am. J. Hypertension (1998) 11:137-146, it was demonstrated that the plasma levels of angiotensin-(1-7) which could be detected in humans suffering from essential hypertension were markedly lower than those which could be detected in normotensive humans.
Hypertension (1996) 28:104-108, showed that angiotensin-(1-7) had an anti-proliferative effect on vascular smooth muscle cells, while Hypertension (1999) 33[part II]:207-211, showed that angiotensin-(1-7) inhibited the proliferation of smooth muscle cells following vascular tissue damage.
In addition to this, angiotensin-(1-7) also exhibited renal effects, such as an increased natriuresis and diuresis, in sodium chloride-loaded, anesthetized normotensive Wistar rats (Am. J. Physiol. (1996) 270:F141-F147).
The compounds of formula (I) which are described herein are potent, nonpeptide agonists of the postulated endothelial angiotensin-(1-7) receptors. They therefore mimic the above-described biological effect, which is directed against angiotensin II, of the peptide hormone angiotensin-(1-7), which effect is to be attributed to the production and/or release of cGMP and NO from the endothelium, without, in this connection, undergoing the rapid metabolic degradation of this hormone. The described compounds of formula (I) are therefore generally suitable for treating and/or preventing diseases where the primary or secondary cause, or at least a primary or secondary component of the cause, is a reduced production and/or release of the vasorelaxing, antithrombotic and cardioprotective messengers cyclic 3xe2x80x2,5xe2x80x2-guanosine monophosphate (cGMP) and nitrogen monoxide (NO). By means of stimulating the production and/or release of these vasorelaxing, antithrombotic and cardioprotective messengers, the described angiotensin-(1-7) receptor agonists of formula (I) are useful as pharmaceuticals for treating and preventing hypertension; cardiac hypertrophy; cardiac insufficiency; coronary heart diseases, such as angina pectoris; and endothelial dysfunction or endothelial damage, for example, as a consequence of atherosclerotic processes, or in connection with diabetes mellitus.
Stimulation of endothelial angiotensin-(1-7) receptors by the agonists of formula (I) causes vasodilatory and organ-protective autacoids to be released. This mechanism differs from that of angiotensin-converting enzyme (ACE) inhibition and AT1 receptor blockade in that it avoids either a decrease in tissue angiotensin (ANG) II (in the case of ACE inhibitors) or effects which are associated with increased ANG II plasma values (in the case of AT1 receptor antagonists) and which are currently not possible to assess.
The compounds of formula (I), and their physiologically tolerated salts, can consequently be used as pharmaceuticals in animals, such as mammals, and particularly in humans. The compounds may be provided on their own, in mixtures with each other, or together with other active compounds, in the form of pharmaceutical preparations. The present invention therefore relates to the use of compounds of formula (I), and/or their physiologically tolerated salts, for producing a medicament for treating or preventing the above mentioned syndromes, and to pharmaceutical preparations which comprise an effective dose of at least one compound of formula (I), and/or of a physiologically tolerated salt thereof, as the active constituent in addition to customary, pharmaceutically acceptable carrier substances and/or auxiliary substances. The pharmaceutical preparations can be intended for enteral or parenteral use and normally comprise from 0.5 to 90% by weight of the compound of formula (I) and/or its physiologically tolerated salts. The quantity of active compound of formula (I) and/or its physiologically tolerated salts in the pharmaceutical preparations is in general from 0.2 to 500 mg, or from 1 to 300 mg.
Pharmaceuticals which can be employed in accordance with the invention and which comprise the compounds of formula (I) and/or their physiologically tolerated salts may be administered enterally, for example orally or rectally. Such administrations may be provided, for example, in the form of pills, tablets, film tablets, sugar-coated tablets, granules, hard and soft gelatin capsules, solutions, such as aqueous, alcoholic or oily solutions, juices, drops, syrups, emulsions or suspensions. The administration can also be effected parenterally, for example subcutaneously, intramuscularly or intravenously in the form of injection solutions or infusion solutions. Examples of other suitable forms of administration include percutaneous or topical administration, for example in the form of ointments, creams, pastes, lotions, gels, sprays, powders, foams, aerosols or solutions, or use in the form of implants.
The pharmaceutical preparations which can be employed in accordance with the invention can be produced using the known standard methods for producing pharmaceutical preparations. For this, one or more compounds of formula (I) and/or their physiologically tolerated salts are brought into a suitable administration form or dosage form together with one or more solid or liquid carrier substances and/or additives or auxiliary substances. If desired, other pharmaceutically active compounds having a therapeutic or prophylactic effect may be combined in the dosage form. Such pharmaceuticals include, for example, those having cardiovascular activity, such as calcium antagonists, ACE inhibitors, AT1 receptor antagonists, NO donors, endothelin receptor antagonists, K channel openers, phosphodiesterase inhibitors, diuretics or xcex1- and xcex2-blockers. The administration form or dosage form can then be used as a pharmaceutical in human medicine or veterinary medicine.
Suitable carrier substances include organic or inorganic substances which are suitable for enteral (for example oral) or parenteral (for example intravenous) administration or topical uses, and which do not react with the active compounds of formula (I). Suitable carriers include, for example, water, vegetable oils, alcohols, such as ethanol, isopropanol or benzyl alcohols, 1,2-propanediol, polyethylene glycols, glycerol triacetate, gelatin, carbohydrates, such as lactose or starch, magnesium stearate, talc, lanolin, Vaseline, acetonitrile, dimethylformamide and dimethylacetamide. Pharmaceutical forms such as tablets, sugar-coated tablets, capsules, solutions, oily or aqueous solutions, syrups, juices or drops, Also, suspensions or emulsions are commonly employed for oral and rectal use. It is also possible to employ mixtures composed of two or more carrier substances. For example, mixtures may be composed of two or more solvents, such as one or more organic solvents together with water. As additives or auxiliary substances, the pharmaceutical preparations may also comprise, for example, stabilizers, wetting agents, emulsifiers, salts (e.g., to influence the osmotic pressure), glidants, preservatives, dyes, flavors and/or aromas and buffering substances. If desired, they may also comprise one or more additional active compounds, for example, one or more vitamins. The compounds of formula (I) and/or their physiologically tolerated salts can also be lyophilized and the resulting lyophilisates can, for example, be used for producing injection preparations. Liposomal preparations are also suitable for topical use.
In connection with the use according to the invention, the dose of the active compound of formula (I), and/or of a physiologically tolerated salt thereof, to be administered depends on the individual case and, as is customary, should be adjusted to the individual circumstances if an optimum effect is to be achieved. Thus, the dose typically depends on the nature and severity of the disease to be treated, the sex, age, weight and individual responsiveness of the human or animal to be treated, the strength and duration of the effect of the compounds employed, whether the therapy or prophylaxis is being performed acutely or chronically, or whether other active compounds are being administered in addition to the compounds of formula (I). In general, when treating the above mentioned syndromes in humans, a dose range of from about 0.1 mg to about 100 mg per kg per day is appropriate for achieving the sought-after effect when being administered to an adult of about 75 kg in weight. A dose range of from 1 to 20 mg per kg per day (in each case mg per kg of body weight) is typical. In this connection, the daily dose can be administered as a single dose or be divided up into several individual doses, for example one, two, three or four individual doses. The daily dose can also be administered continuously. Where appropriate, it can be necessary, depending on the individual response, to diverge, either upwards or downwards, from the specified daily dose. Pharmaceutical preparations normally comprise from 0.2 to 500 mg, or from 1 to 300 mg, of active compound of formula (I) and/or its physiologically tolerated salts.
The following assays (tests 1 and 2) demonstrate the affinity of the compounds of formula (I) for angiotensin-(1-7)-binding sites and their agonistic properties on endothelial cells:
Test 1: Binding assay
The affinity of the compounds of formula (I) for angiotensin-(1-7) receptors was measured by means of ligand displacement experiments which were carried out on preparations of membranes obtained from primary bovine aorta endothelial cells, as are also described, for example, in Hypertension (1997) 29[part 2]:388-393, herein incorporated by reference.
a) Membrane preparation:
After endothelial cells had been isolated from bovine aortas (test 1, a)), the cells were cultured in 75 cm2 culture flasks (Becton Dickinson, Heidelberg) until they had reached confluence. After that, the cells were taken up with ice-cold phosphate-NaCl-EDTA buffer (50 mmol/ of NaHPO42 0.15 mol of NaCl/L, 5 mmol of EDTA/L, pH 7.2), detached using a rubber scraper and centrifuged (1500xc3x97g, 5 min). The resulting cell pellet was frozen (xe2x88x9280xc2x0 C.) for subsequent membrane preparation.
The thawed cell pellet was homogenized in ice-cold phosphate-NaCl-EDTA buffer (glass/teflon Potter, 1000 rpm, 10 strokes). The membranes were isolated by subsequently centrifuging (30000xc3x97g, 20 min) the cell homogenate. The cell pellet which was obtained in this way was resuspended in modified HEPES buffer (10 nmol of HEPES/L, 0.1 mol of NaCl/L, 5 mmol of MgCl2/L, pH 7.4) in the added presence of 0.2% bovine serum albumin and a cocktail of protease inhibitors (Complete(trademark), Boehringer Mannheim). After a protein determination had been subsequently carried out on the membrane suspension (using the Lowry method), the suspension was immediately used for the ligand binding experiment.
b) Binding experiments:
The experiments were carried out on 96-well Opak plates, which are equipped with Durapore filters (0.65 xcexcm pore size; Millipore, Eschborn). Before beginning the experiment, the filters were pretreated for 30 min with 1% bovine serum albumin in order to minimize nonspecific binding of the radioactive ligand and the cold substances to the filter material. The incubation was carried out in a total volume of 200 xcexcl: 50 xcexcl of 125I-ANG-(1-7), 20 xcexcl of cold, nonradioactive ANG-(1-7) or test substances of formula (I), 30 xcexcl of buffer and 100 xcexcl of membranes (20 xcexcg of protein). The binding reaction was started by adding the radioactive ligand. The samples were incubated at room temperature for 45 min while being continuously shaken. The binding reaction was terminated by means of vacuum filtration (xe2x88x9220 kPa vacuum; Multiscreen filtration system, Millipore, Eschborn). In order to completely remove the free radioactivity, which was not membrane-bound, the filters were washed twice under vacuum with 250 xcexcl of ice-cold phosphate-NaCl-EDTA buffer (50 mmol of NaHPO4/L, 0.15 mol of NaCl/L, 5 mmol/L EDTA, pH 7.2) and then dried. The radioactive content on the dried filters was determined using a gamma counter.
For the competition experiments (determination of xe2x80x9cindividual valuesxe2x80x9d or IC50 values), a concentration of from 7.5 to 10 nmol of 125I-ANG-(1-7)/L (specific activity, 1500-2100 mCi/mg) was employed, with and without increasing concentrations of the test substances of formula (I). The nonspecific binding was in each case measured in the presence of 10 xcexcmol of nonradioactive ANG-(1-7)/L.
c) Results:
These values, which are listed by way of example, for the compounds of Examples 4 and 9, infra, demonstrate the high affinity of compounds of formula (I) for angiotensin-(1-7) receptors on endothelial cells. In this connection, the compounds of formula (I) exhibit no affinity, or only a negligible ( greater than 10xe2x88x926 M) affinity, for ANG II receptors of the AT1 and AT2 types.
Test 2: Functional assay:
The stimulatory effect of the compounds of formula (I) on the production of intracellular cGMP, which is a marker for the production and release of NO in endothelial cells, was measured on primarily cultivated endothelial cells derived from bovine aortas, as described, for example, in J. Pharmacol. Exp. Ther. (1992) 262:729-733, herein incorporated by reference.
a) Cell cultures:
After having been enzymatically digested (dispase II; Boehringer, Mannheim) from the bovine aorta, the endothelial cells were taken up in culture medium (Dulbecco""s modified Eagle""s Ham""s F 12 medium 1:1 containing penicillin (10 U/L), streptomycin (10 ug/L), L-glutamine (1 mmol/L), glutathione and L-(+)-ascorbic acid (in each case 5 mg/L) and heat-inactivated fetal calf serum (20%)), washed once (centrifugation at 170xc3x97g, 10 min) and resuspended in culture medium. This cell suspension was seeded in 6-well plates (Nunc Intermed, Wiesbaden) (xcx9c250 xcexcg of protein or 3xc3x9710xe2x88x925 cells per well), with the wells then being filled with culture medium and the plates kept at 37xc2x0 C. in an incubator which was moistened and gassed with 95% O2-5% CO2.
b) cGMP determinations:
After confluence had been reached (6-8 days after seeding), the culture medium was removed and the cell monolayer was washed twice with warm HEPES/Tyrode""s solution. After that, the cells were preincubated, at 37xc2x0 C. for 15 min, in HEPES/Tyrode""s solution containing IBMX (3-isobutyl-1-methylxanthine, 10xe2x88x924 mol/L, Serva, Heidelberg). The incubation was started by adding SOD (bovine erythrocyte superoxide dismutase, 3xc3x9710xe2x88x927 mol/L, Serva, Heidelberg) and the test substances of formula (I) at the given concentrations. After the appropriate incubation time, the incubation medium was aspirated and the cells which remained behind were immediately extracted in 1 N formic acid/acetone (v/v, 15:85) and scraped off. The resulting suspension was ultrasonicated (10 sec) and then centrifuged down (3000xc3x97g, 10 min). For the purpose of determining cGMP by means of radioimmunoassay (New England Nuclear, Boston, Mass.), the supernatant was lyophilized and the lyophilisate taken up in sodium acetate buffer (0.05 mol/L; pH 6.2). The content (pmol) of intracellular cGMP was related to mg of cell protein.
c) Results:
The listed values for the compounds described in Examples 4 and 9, infra, which compounds are taken as being representative of the claimed compounds, demonstrate the agonistic effect of the compounds of formula (I) on angiotensin-(1-7) receptors.
At the same time, this effect of the compounds from Examples 4 and 9, infra, on the production of cGMP, as a marker for the synthesis and release of NO, is not influenced by preincubation with an angiotensin II receptor antagonist of either the AT1 subtype, such as EXP3174, or of the AT2 subtype, such as PD 123,319. By contrast, the stimulatory effect of the compounds from Examples 4 and 9 on cGMP is inhibited by preincubation with a selective antagonist of the angiotensin-(1-7) receptors, i.e. [D-Ala7]-angiotensin-(1-7), which is described, for example, in Brain Res. Bull. (1994) 35:293-298, herein incorporated by reference, thereby demonstrating the specificity of this functional effect.
List of abbreviations:
abs. absolute
cGMP cyclic guanosine monophosphate
CH2Cl2 dichloromethane
DCI desorption chemical ionization
DMF N,N-dimethylformamide
EA ethyl acetate
ESI electron spray ionization
FAB fast atom bombardment
M.p. melting point
M.S. mass spectrometry
sat. saturated
h hour(s)
min. minute(s)
NO nitrogen monoxide
RT room temperature
THF tetrahydrofuran